We have developed a powerful new Fluorescent Speckle Microscopy (FSM) method that utilizes epi-fluorescence microscopy to analyze the movement, assembly, and disassembly of macromolecular structures in vivo and in vitro. Initially, we devised this method for studying the dynamics of the cytoskeletal polymers, microtubules and actin filaments in cell locomotion and division. In FSM, structures are assembled from a low fraction of fluorescently labeled subunits together with unlabeled subunits and imaged with high-resolution optics and a sensitive, low-noise digital camera. FSM is achieved in living cells by microinjection or expression of a low amount of fluorophore-conjugated subunits. Stochastic variations in the number of fluorescent subunits per resolution-limited image region results in a "speckled" appearance of the assembled structure. In time-lapse FSM, movement and changes in speckle intensity act as reporters for structure translocation, assembly and disassembly. FSM is fast becoming the method of choice for studies of cytoskeleton dynamics. However, FSM requires further development to reach its full potential for biomedical research. Focus drifts during imaging are a major source of FSM artifact, and the versatility of FSM for various fluorescence microscope modes has to be demonstrated. In addition, the analysis of time-lapse FSM images are currently done by hand, which is slow, incomplete, and prone to error. This must be replaced by automated, quantitative FSM image analysis. Finally, FSM image generation and analysis techniques have to be broadened to general biomacromolecular assemblies. To achieve these goals, we have the following Specific Aims: 1. Hardware Development: To develop focus-stabilized FSM and multi-spectral Total Internal Reflection FSM (TIR-FSM) for studying protein dynamics in vitro and on ventral cell surfaces in vivo. 2. Software Development: To develop software for automatic quantitative analysis of protein dynamics in time-lapse FSM images. 3. Applications: To use multi-spectral FSM, TIR-FSM, and analysis software for studying other biologically important systems besides cytoskeletal polymers. These advances will allow FSM to become a powerful tool for studying molecular dynamics in cell biology, neurobiology, biotechnology, and material science.